Methods of changing one or more nucleotides in a nucleic acid sequence are useful in several applications, including the identification and alteration of gene function in an organism. Such site-directed gene manipulation has been moderately successful via homologous recombination in animal models, and particularly, in mammalian cell cultures. See, e.g., Yoon, et al., Proc. Natl. Acad. Sci. USA, 93:2071–2076 (1996).
Unfortunately, the success of conventional homologous recombination in animal models has been limited in plant cells due to the low frequency of homologous recombination. See, Zhu et al., Proc. Natl. Acad. Sci. 96:8768–8773 (1999). Moreover, this method also suffers from complications of random insertion in the absence of correct sequence homology. See, Capecchi, M. R., Science 244:1288–1292 (1989). Triple-helix forming oligonucleotides coupled to cross linking agents have been used with limited success to alter DNA sequences. However, the utility of this approach is constrained by the absolute necessity that the target DNA sequence must consist of homopyrimidine or homopurine stretches in the target DNA sequences.
Another method for gene targeting involves the use of chimeric DNA/RNA oligonucleotides to introduce single-point mutations in certain nucleotide sequences. Such mutations induced by chimeric DNA/RNA oligonucleotides generally have involved alteration of 1–2 base pairs in the target site, and is suitable for many applications, such as site specific mutagenesis, gene knockouts, and allelic replacements. For example, tyrosinase is an essential enzyme in melanin synthesis, produced by melanocytes, and sufficient for pigmentation change in vitro and in vivo. Melanocytes from albino mice contain a homozygous point mutation (TGT→TCT) in the tyrosinase gene. This results in an amino acid sequence change from Cys to Ser at amino acid 85 of the mature tryosinase. This single amino acid change is responsible for the complete inactivation of tyrosinase and the resultant absence of pigmentation. Thus, correction of the point mutation in tyrosinase gene results in the restoration of the enzyme activity and in changes of the pigmentation of the cell.
This ability to correct single point mutations has enabled the creation of therapeutic strategies for the treatment of genetic diseases not otherwise available. For example, sickle cell anemia is the classic prototype of a hereditary hemoglobinpathy resulting from a point mutation in the β-globin gene. The single point correction of an A- to -T mutation within the β-globin results in the change from a mutant valine to the normal glutamic acid residue. Correction of the mutant allele through a gene conversion mechanism provides a means for gene therapy that avoids the the attendant problems associated with current therapies such as transduction methods based on retro- and adenovirus vectors. See, e.g., Cole-Strauss, et al. Science, 273:1386–1389 (1996).
It may be possible to use such chimeric DNA/RNA oligonucleotides to: (1) create cell and animal disease models for drug target validation (2) produce novel plants with desired input traits, e.g., heat, cold and salt resistance, and output traits, e.g., higher fiber content and nutritional value (3) correct small genetic mutations that cause disease (4) alter normal genes and thereby modulate disease-relevant pathways and (5) optimize other industrial applications such as fermentation. See, http://www.valigen.net/tech.htm.
Chimeric mutational vectors (CMVs) having non-natural nucleotides are described in U.S. Pat. Nos. 5,731,181 and 5,795,972. The CMVs are said to comprise two complimentary oligoncleobase strands, wherein the oligonucleobases are either ribo-type (i.e., having a 2′ hydroxyl) or 2′-deoxyribo-type. At least three contiguous bases of one strand are ribo-type nucleobases, preferably nuclease resistant nucleobases. The patents state that the nucleobases of a chain of a CMV can be any nucleobase now known or to be developed that hybridizes by Watson-Crick base pairing to DNA. CMVs have been shown to effect single point mutations in wild type human liver/bone/kidney episomal alkaline phosphatase.
While these strategies have been successful in animal models, little is known about the use of single point gene modification or the introduction of CMVs in plant systems. As noted above, the applicability of site-specific gene modification has wide reaching utilities in the agricultural industry, including the development of herbicide- and disease-resistant plants. For such applications, heritablity of gene modifications and stable transmission of modified genetic traits to plant progeny is a necessity. While chimeric DNA/RNA oligonucleotides have been used to engineer herbicide-resistant maize plants without integrating foreign genes or regulatory sequences into the plants, the frequency rates in plants are approximately three orders of magnitude lower than those reported for chimeric gene modification in animal systems. See, Zhu et al., Nature Biotechnology, 18:555–558 (2000). Studies suggest this lower success rate may be due to the differences in efficiencies of homologous pairing, strand transfer, or mismatch repair between mammalian and plant cells. Thus, a need remains for addressing these lower frequencies of gene modification in plant systems. Likewise, there exists a need to improve upon the homologous pairing, strand transfer, or mismatch repair efficiencies currently attainable.
Even in animal models, the success rate of current chimeric RNA-DNA techniques converting single point mutations is quite low, hovering around 13–30%. See generally, Kren et al, Nature Medicine 4:285–290 (1998). Indeed, the low frequency of specificity remains a serious limitation in the realm of therapeutics. Severe difficulties arise under conditions where the single point mutation is inaccessible due to intrastrand folding and intermolecular conformational stabilities.
Recently, oligonucleotide analogues have been developed to investigate the conformational transition that occurs when oligonucleotides hybridize to a target sequence, from the relatively random coil structure of the single stranded state to the ordered structure of the duplex state. For example, conformationally restricted oligonucleotide analogues that include locked nucleoside analogues (LNAs) are described in PCT WO 99/14226. As an example, bicyclic LNAs contain nucleosides with a 2′-O-4′-C methylene bridge. Other bicyclic and tricyclic LNAs are described therein and are incorporated by reference, including any drawings. This conformational and steric hindrance is believed to inhibit nuclease attack of the LNAs, thereby resulting in an increased thermal stability of duplexes formed between LNAs and complementary DNA or RNA. To date, while LNAs have been developed as blocking agents for translation and transcription in vitro and in vivo, as sequence specific inhibitors such as PCR clamping, as well as in various antisense therapies, the unique capabilities of LNA have not been harnessed for use in the repair of single point mutations. See. e.g., www.proligo.com.
Thus, there still remains a need for additional methods of replacing nucleotides in target nucleic acid sequences for a wide variety of uses relating to the identification and alteration of gene function in an organism. Accordingly, a need also exists for an improved method of introducing a single point mutation into cells with an increased efficiency than is currently attainable.